The present invention relates to the fields of genetic engineering, biochemistry, and plant biology.
N-phosphonomethylglycine has the following structure: ##STR1## This molecule is an acid, which can dissociate in aqueous solution to form phytotoxicant anions. Several anionic forms are known. As used herein, the name "glyphosate" refers to the acid and its anions. A mixture containing glyphosate as the active ingredient, formulated as its isopropylamine salt, is sold as a herbicide by Monsanto Company under the trademark ROUNDUP.RTM.. Numerous other salts also have herbicidal properties, as exemplified by U.S. Pat. No. 3,799,758 (Franz 1974) and various other patents. Compositions comprising N-phosphonomethylglycine and salt-forming cations which increase the solubility of the N-phosphonomethylglycine in water are preferred.
Those skilled in the art recognize that the scientific literature contains numerous papers suggesting several modes of action for inhibition of plant growth by glyphosate. One proposed mode suggests that glyphosate inhibits an enzyme called 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS); see, e.g., Amrhein 1980, Steinrucken 1980, Mousdale 1984, and Rubin 1982 (note: a complete list of references is contained below, after the Examples). The EPSPS enzyme reportedly catalyzes the conversion of shikimate-3-phosphate into 5-enolpyruvyl-shikimate-3-phosphate, an intermediate in the biochemical pathway for creating three essential aromatic amino acids (tyrosine, phenylalanine, and tryptophan); see, e.g., Mousdale 1984. Rogers 1983 reports that overproduction of EPSPS in E. coli contributes to glyphosate resistance in those cells.
At least one researcher has attempted to create glyphosate-resistant bacterial cells by manipulating a bacterial gene which encodes an EPSPS enzyme. As described in U.S. Pat. No. 4,535,060 (Comai; assigned to Calgene, Inc.; filing date Jan. 5, 1983) and in Comai 1983, a culture of Salmonella bacteria was contacted with a mutagen (ethyl methanesulfonate). The bacteria were screened for glyphosate resistance, and a relatively resistant culture was selected. This culture was analyzed, and determined to have a mutant form of EPSPS with a substituted amino acid, as reported in Stalker 1985. U.S. Pat. No. 4,535,060 suggested that the mutant EPSPS gene could be inserted into plant cells to create glyphosate-resistant (Gly.sup.R) plant cells. In addition, it has been reported that glyphosate tolerant plant cells can be selected which overproduce EPSPS in the presence of low levels of glyphosate (Nafziger et al, 1984 and Smart et al, 1985). However, none of the experiments have demonstrated that such a method would be efficacious in differentiated plants.
After the filing date of U.S. Pat. No. 4,535,060, methods and vectors were described which could be used to insert foreign genes into plant cells (see, e.g., Fraley 1983, Herrera-Estrella 1983, Bevan 1983, and PCT applications WO No. 84/02919 and 02920). In PCT application WO No. 84/02913, methods were also described for creating chimeric genes having bacterial EPSPS coding sequences controlled by regulatory sequences derived from genes which are active in plant cells. Using these vectors and methodology, bacterial genes such as the mutant Salmonella EPSPS gene mentioned above can be manipulated and expressed in plant cells.
The object of this invention is to provide a method of genetically transforming plant cells which causes the cells and plants regenerated therefrom to become resistant to glyphosate and the herbicidal salts thereof.